Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 8000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The mortality rate is roughly that of cancer. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The gene for pol gamma is called POLG. To date there are more than 60 pathogenic mutations in POLG. POLG, the gene for the catalytic subunit of pol gamma, is one of several nuclear genes that are associated with mitochondrial DNA depletion or deletion disorders. Dysfunction of the pol g has been associated with such disorders as: PEO, parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). PEO is a mitochondrial disorder associated with mtDNA deletions and point mutations. PEO is characterized by late onset (between 18 and 40 years of age) bilateral ptosis and progressive weakening of the external eye muscle, resulting in blepharoptosis and ophthalmoparesis, proximal muscle weakness and wasting as well as exercise intolerance. The disease is often accompanied by cataract, hypogonadism, dysphagia, hearing loss and may, within several years, lead to development of neuromuscular problems. Neurological problems may include depression or avoidant personality. Skeletal muscles of PEO patients present red ragged fibers and lowered activity of respiratory chain enzymes. PEO can be inherited in an autosomal dominant or recessive manner. Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder that affects children and young adults. It is a devastating disease characterized by psychomotor retardation, hepatic failure and intractable seizures, as well as tissue-specific mtDNA depletion. Alpers syndrome is 100% fatal with no cure available. This syndrome has been exclusively associated with mutations in POLG. Carriers for this disease are 1:250 with presentation occurring 1:100,000 to 250,000. The first mutation associated with Alpers syndrome in POLG was described only last. Since this publication we and others have identified over 15 pathogenic POLG mutations that cause Alpers in over 20 probands. Ataxia/neuropathy resulting from mutations in POLG is an autosomal recessive disorder affecting patients in their mid-teens to later years usually resulting in premature death. The disease is accompanied mainly by mtDNA deletions. The ataxia usually occurs in combination with various central nervous system features including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and evidence of neuronal loss in discrete gray nuclei. Among the nearly 60 disease mutations in the POLG gene, the A467T substitution is the most common and has been found in 0.6% of the Belgian population. The A467T mutation is associated with a wide range of mitochondrial disorders, including Alpers syndrome, juvenile spino-cerebellar ataxia-epilepsy syndrome, and progressive external ophthalmoplegia, each with vastly different clinical presentations, tissue specificities, and ages of onset. We overproduced the A467T mutant pol gamma and found the enzyme possesses only 4% of wild-type DNA polymerase activity, and the catalytic defect is manifest primarily through a 6-fold reduction in kcat with minimal effect on exonuclease function. The A467T defect disrupts the balance of exonuclease to polymerase activity in the enzyme resulting in uncontrolled exonucleolytic degradation of DNA. Human pol gamma requires association of a 55 kDa accessory subunit for enhanced DNA binding and highly processive DNA synthesis. However, the A467T mutant enzyme failed to interact with, and was not stimulated by, the accessory subunit, as judged by processivity, heat inactivation, and N-ethylmaleimide protection assays in vitro. Thermolysin digestion and immunoprecipitation experiments further indicate weak association of the subunits for A467T pol gamma. This is the first example of a mutation in POLG that disrupts physical association of the pol gamma subunits. We propose that reduced polymerase activity and loss of accessory subunit interaction are responsible for the depletion and deletion of mitochondrial DNA observed in patients with this POLG mutation. In patients with Alpers syndrome we identified five new POLG mutations (F749S, R852C, T914P, L966R, and L1173fsX) in five unrelated kindreds, and summarize 14 allelic combinations of POLG mutations now known to cause Alpers syndrome from 20 probands published to date. In a sequential series of 15 DNA samples from probands with Alpers syndrome, we sequenced 379,827 nucleotides of POLG DNA, and identified two pathogenic mutations in each of 13 samples. The most common Alpers-causing mutation is the A467T substitution in the linker region of the pol gamma protein. All patients had at least one mutation in the linker region, providing a simple molecular diagnostic screen for this disorder. Our results provide the first molecular epidemiologic evidence for an important role of the linker region in the development of the Alpers phenotype. Additionally, we identified several trends of mutations involved with the A467T mutations, which allows for a molecular diagnosis of POLG diseases involving A467T.